Flow cytometry is a field which has existed for many years. Basically, flow cytometer systems act to position small amounts of a substance within a sheath fluid. Through hydrodynamic focusing and laminar flow, the substance is split into individual particles, cells, or the like. In many applications, sheath fluid together with its entrained substance exits a nozzle in a jet and free falls or is channeled in an optically transparent pathway for analysis. The sheath fluid may form droplets encapsulating individual particles which are separated and collected based upon assignment of differentiated particle characteristics.
This type of analysis requires uniform conditions within the jet, very precise timing, and consistent comparative parameters incident to the entrained substances to separate such substances accurately. In addition, there is a coincident commercial and public sector demand for higher speed flow cytometry, the need to differentiate substances based on more complex and multiple parameter analysis, and for higher purity separation(s). Unfortunately, variation in equipment operation, sheath fluid stream dynamics, or observed particle characteristics still exists and are exacerbated by increasing the speed at which entrained substances are carried in the jet. As such, there is a need to compensate for such variations to provide for accurate analysis and separation of the substances entrained in the sheath fluid stream.
An overview of some attempts to understand and react to fluid stream and droplet dynamics can be seen in U.S. Pat. Nos. 4,317,520, 4,318,480, 4,318,481, 4,318,482, 4,318,483, and 4,325,483, each hereby incorporated by reference herein. As these explain, traditionally the approach has been to assess the signals and act directly upon such information. Some of the practical problems which have also been recognized is the fact that only a limited amount of space and time exists within which to conduct sensing and analysis. As Japanese Patent 2024535 also recognizes with respect to the sensing system alone, it may be desirable to have an optical system which is as small as possible.
As can be understood, a substantial problem can be that the data generated from an occurrence must be sensed and reacted upon in an extremely short period of time. Given the speed of microprocessors and the like, this might, at first glance, appear to be readily achievable. The challenge for this unique flow cytometry situation is that original or raw signal data can be sub-optimal and even unusable. As such, if it is to be used, it must be further processed in order to accomplish further analysis or decision making. This processing can be complex and can require more processing speed and power than is available not just with typical commercial systems, but even with today's highest-speed computer systems. Further, as the desire for higher processing frequencies is pursued, problems can be compounded. An example of the extremes to which speed has been taken is shown in U.S. Pat. No. 4,361,400, hereby incorporated by reference herein, where droplet formation frequencies in the range of 300 to 800 kilohertz had been achieved. Most practical droplet flow cytometers operate in the range of 10 to 50 kHz. Although speed of analysis problems have been known for years, prior to the present invention it has apparently been an accepted attitude that digital analysis in the flow cytometry context could not be achieved. This invention proves this expectation to be untrue. As a result of the present invention, droplet formation speeds in the 50–100–200 or higher kHz ranges are now possible with adequate data compensation and the like.
At any of these speeds, however, there appears to have been an expectation that analog analysis was the only practical way to achieve analysis of and to compensate for fluid dynamics, particle characteristics, equipment variance, and the like. To some degree, these expectations have been so prevalent that quality control, good manufacturing practices, regulatory approval, and other concerns have been set aside, diminished, or even compromised. The previously existing technology governing the practices of those in this field.
Another significant problem associated with conventional analysis and compensation of variables in flow cytometry can be the preservation of original signal data from an occurrence incident to the fluid stream prior to subsequent processing steps. It may not have been possible to preserve or store original signal data until now due to the short amount of time in which to analyze or compensate the original signal. As such, all or part of the original or raw signal data may have been sacrificed to increase the efficiency of analysis or provide feed back compensation events. The practice of discarding original raw data may prevent re-analysis of the data to improve quality control, to establish good manufacturing practices, and attain procedural thresholds for certain regulatory or statutory requirements.
Yet another problem with conventional analysis may be the inability to process high speed serial occurrences, to compensate multiple parameters, to perform complex operations, to provide transformation compensation of original data, or to apply compensated parameters. Conventional analysis can be limited by the amount of information that can be processed and returned in between serial events which can occur at a rates of at least 10,000 per second.
A first aspect of this inability can be associated with the nature of conventional signal processors used with flow cytometry. Conventional flow cytometer signal processors, often because they are analog, are not capable of dealing with large amounts of signal information, cannot perform operations on low quality signal information, cannot practically accomplish complex transformation operations (such as those which use algebraic expressions or structure), or they perform only reflexive feed back operations rather than serial or multi-variant analysis followed by subsequent parameter compensation.
A second aspect of this inability can be associated with the infrastructure of conventional data handling. In part, conventional infrastructure may not deal with how the streams of information are allocated, aligned, and coordinated. Conventional processing of flow cytometer information from occurrences incident to the fluid stream are traditionally handled as isolated feedback loops. As such, it can become increasingly difficult to synchronize various aspects of flow cytometer operation as the number of feed back loops increases. Moreover, these feed back loops may be completely uncoupled. For example, stream parameters, such as droplet break off location, may be completely uncoupled from the differential analysis of and separation of particles within the fluid stream being compensated.
A third aspect of this inability may be lack of symmetry reduction in the application of transformed data. Again, analog analysis can prevent or minimize symmetry reduction in the complex analysis of serial occurrences or parallel multivariant analysis. The lack of symmetry reduction or the inability to apply symmetry reduction to analysis terms may increase execution time.
As mentioned above, there has been a long felt but unsatisfied need for apparatus and methods which permit complex signal transformation, and use of compensated parameters resulting from complex signal transformation, real time analysis using compensated parameters, or storage of original signal data generated incident to the fluid stream, instrument variance, or environmental variance. The present invention addresses each of the above-mentioned problems with a practical solution. To some extent, it is apparent that solutions have not been achieved because those skilled in the art seem to have taken a direction which was away from the technical direction pursued in the present invention. This may have been the result of the fact that those skilled in the art did not truly appreciate the nature of the problem or it may have been the result of the fact that those skilled in the art were misled by some of the presumptions and assumptions with respect to the type of systems which could be considered. The present invention uses digital signal processing (DSP) technology to structure information from occurrences incident to flow cytometer operation, and to perform complex transformation, compensation, or analysis operations to achieve this long sought goal.